Translation initiation is a critical step in gene expression. The majority of eubacterial mRNAs interact with ribosomes via complementarity between a region on the ribosomal RNA and a sequence contained in a leader region upstream of the mRNA coding sequence. However, several mRNAs from diverse systems (e.g., eubacteria, archaebacteria, and human mitochondria) lack any upstream region but are still translated. One objective of this proposal is a general demonstration that removal of mRNA leader regions does not prevent their accurate translation by ribosomes from the eubacterial genera Streptomyces and Escherichia. Various alterations will be introduced to the 5' end of a leaderless mRNA to characterize the constraints for AUG codon placement in translation initiation and reading frame specification. A combination of genetic, biochemical, and molecular biological techniques will be used to examine in vivo and in vitro interactions between ribosomes and leaderless mRNA. The mechanistic details of these interactions will be probed by a search for cellular factors (e.g., ribosomal proteins, translation initiation factors) that bind leaderless mRNA to ribosomes or function to specify the correct reading frame. Also, conditions will be established to inhibit translation by conventionally leadered mRNA in an effort to identify other cellular proteins that might be translated independent of interactions between leader regions and ribosomal RNA. The experiments in this proposal address fundamental aspects of translation and will contribute to our general understanding of translation initiation in a variety of biological systems.